冬季太湖表层底泥产毒蓝藻群落结构和种群丰度

应用荧光定量PCR对冬季太湖不同湖区底泥表面有毒微囊藻和总微囊藻种群丰度进行调查,同时基于PCR-DGGE技术对底泥中有毒微囊藻群落结构进行分析。结果表明:微囊藻在太湖底泥表面分布广泛,所有采样点都检测到有毒微囊藻存在,且不同湖区有毒微囊藻和总微囊藻种群丰度存在显著差异,有毒微囊藻和微囊藻基因型丰度范围分别为1.23×10⁴-3.75×10⁶拷贝数/g干重和2.56×10⁴-1.07×10⁷ 拷贝数/g干重,有毒微囊藻与微囊种群丰度的比例为4.8％-35.2％;DGGE指纹图谱显示,冬季太湖不同湖区表层底泥中有毒微囊藻群落结构相似性较高,相似性系数为70.2％-96.0％。虽然不同湖区基因型组成存在差异,但所有样品中占优势的基因型是一致的。同时发现,优势基因型所占的比例与样品的香农多样性指数呈负相关。序列分析表明,mcyA序列长度为291bp,序列相似性超过97％。综合定量PCR结果和底泥中叶绿素a和藻蓝素浓度的测定结果,可以得出2010年冬季太湖蓝藻越冬主要集中在梅梁湾、竺山湾、贡湖湾和湖心。通过建立荧光定量PCR分析方法,为研究湖泊底泥中蓝藻种群丰度动态变化奠定了基础。     

有毒和无毒微囊藻对大型溞生长和繁殖的影响

近年来,随着工农业的快速发展和人们生活水平提高,一些城市生活污水和工农业废水流入江河、湖泊等淡水水体中,加剧了水体的富营养化和污染,导致蓝藻过度生长繁殖而形成水华,其中最常见的是微囊藻水华[1,2].     

夏季蓝藻水华期间太湖河口区和敞水区纤毛虫群落组成及水平分布

在太湖北部主要河口区及敞水区域采集纤毛虫样品, 并用定量蛋白银法进行分析。河口区设10个采样点, 敞水区设14个采样点。调查中共检出3纲15目60属的105种纤毛虫, 种类最多的是钩刺目（21种）, 其次是寡毛目（20种）。纤毛虫优势种及其在河口区和敞水区占总丰度比例分别为： 寡毛目的双叉弹跳虫Halteria bifurcate Tamar （12.3%、18.1%）、大弹跳虫H. grandinella Dujardin （12.3%、10.9%）、短列裂隙虫Rimostrombidium brachykinetum Krainer （8.0%、13.4%）、圆筒状似铃壳虫Tintinnopsis cylindrata Kofoid Campbell （11.8%、4.5%）、盾纤目的银灰膜袋虫Cyclidium glaucoma Mller （3.1%、10.8%）。其他常见种类还有： 前口目的趣尾毛虫Urotricha farcta Claparde Lachmann、寡毛目的杯铃壳虫Codonella cratera Leidy、小裂隙虫R. humile Penard、奇异急游虫Strombidium mirabile Penard、小筒壳虫Tintinnidium pusillum Entz、缘毛目的水生钟虫复合种Vorticella aquadulcis complex和钟形钟虫V. campanula Ehrenberg。河口区和敞水区纤毛虫平均丰度分别为31407 cells/L（范围1 600-80 900 cells/L）和18618 cells/L（范围1225 -36000 cells/L）, 平均生物量分别为1322.6 g/L（范围44.6-3119.7 g/L）和543.6 g/L（范围44.0-1570.4 g/L）。两个区域的优势类群相似, 均以寡毛目、前口目、盾纤目和缘毛目为主。但河口区纤毛虫生物多样性（种类丰富度、Simpson多样性指数、Margalef多样性指数、Shannon多样性指数）显著高于敞水区的（P0.05）。统计分析发现, 食藻种类与叶绿素a含量之间无显著相关, 而食菌种类的丰度和叶绿素a含量呈显著正相关关系（P0.001）。由此推测, 纤毛虫群落结构的空间差异可能与水华暴发程度有较大关系。     

滇池水华蓝藻铜锈微囊藻和绿色微囊藻的生长生理特性及毒素分析

从滇池分离纯化了两种常见水华微囊藻即铜锈微囊藻和绿色微囊藻。在常规培养条件下,两种藻类在对数生长期的生长速率μ值分别为0.61和0.63;早期生长的抑制光强不大于100μmol m~(-2)s~(-1)。铜锈微囊藻主要产生3种微囊藻毒素:MYCST-RR,MYCST-YR和MYCST-LR,绿色微囊藻产生的主要微囊藻毒素为[Dha~7]-MYCST-RR,和[Dha~7]-MYCST-LR,另含有少量的[Dha~7]-MYCST-YR。在低光强15μE m~(-2)s~(-1)时,毒素含量每毫克干重细胞达到3.127μg微囊藻毒素,当光强达到100μE m~(-2)s~(-1)时,毒素含量降低到每毫克干重细胞1.971μg;光强对毒素形成的影响受到温度的调节,而温度对毒素形成的影响不大。探讨了两种微囊藻细胞在不同光照强度下叶绿素荧光比值Fv/Fm的变化,此比值的变化可以间接反映细胞受外界光照强度抑制程度。     

有毒铜绿微囊藻胁迫下三角帆蚌消化系统的扫描电镜观察

通过观察暴露于不同浓度有毒铜绿微囊藻(Microcystis aeruginosa)下的三角帆蚌(Hyriopsis cumingii)的胃、前肠、中肠以及晶杆体等消化系统的扫描电镜切片,了解有毒铜绿微囊藻对三角帆蚌消化系统超显微结构的影响。结果显示,当三角帆蚌在浓度为1×107cell/L的有毒铜绿微囊藻环境下,其晶杆体随时间推移而逐渐溶解。在低浓度的铜绿微囊藻环境下,尽管其会继续滤食,但有毒铜绿微囊藻会对其消化系统造成破坏,随着接触时间的增长,造成其肠道内绒毛、纤毛数量减少,长度降低,从而降低其消化效率,对其生长造成影响。     

应用RT-qPCR技术定量检测湖泊水体中蓝藻方法的比较

利用RT-qPCR技术建立了对湖泊水体中的微囊藻和蓝藻的SYBR Green Ⅰ荧光定量PCR检测方法,在所建立的方法中,对以微囊藻藻蓝蛋白基因、蓝藻16S rRNA基因、微囊藻16S rRNA基因分别作为RT-qPCR检测的目的基因所得结果进行了比较,并对实验室培养的微囊藻和太湖的环境样品进行了检测。结果表明,用藻蓝蛋白基因作为检测目的基因,以M.aeruginosa PCC 7806基因组DNA作为标准品的测定方法与显微镜计数的结果有较好的相关性和一致性,并具有简便、快速、特异性高的特点,可以满足检测的要求。     

荧光原位杂交技术和流式细胞仪用于环境样品中产毒及非产毒微囊藻的定量监测

全球范围内,高频次、大范围暴发的蓝藻水华对淡水水体环境造成严重影响.微囊藻因其在生长特别是衰亡过程中向水体释放微囊藻毒素而威胁人类健康.因此,分析其产毒株及非产毒株在环境样品中的组成,建立产毒蓝藻的预报及评价体系显得极为重要.本文采用荧光原位杂交技术结合流式细胞技术实现对环境样品中产毒藻株的鉴别与定量.针对目标基因mcyA设计的、以地高辛标记的双链DNA探针可有效应用于产毒微囊藻FACHB905和PCC7806的鉴别.分别对来自滇池、太湖和关桥的11个样品进行分析显示,该方法与传统的形态学鉴定及PCR方法有较好的匹配.荧光原位杂交技术与流式细胞相结合可有效鉴别产毒与非产毒微囊藻,尤其可以对野外样品中产毒与非产毒藻株进行简便、可视化地鉴别,从而达到对产毒微囊藻水华早期预警的目的.     

应用高通量实时荧光定量PCR方法筛选FL细胞对低剂量微囊藻毒素LR暴露的应答基因

微囊藻毒素是蓝藻的一些属产生的单环七肽,在发生水华的水体中普遍存在[1]。含有微囊藻毒素的水华能引起野生动物、鱼类、家畜、家禽等中毒和死亡,也对人类健康构成严重威胁[2,3]。流行病学调查发现,人群原发性肝癌、大肠癌发病率与饮水源中的微囊藻毒素有关[4]。其中微囊藻毒素LR是微囊藻毒素中最常见的一种,因其高急性毒性,强促癌活性而受到广泛的关注。有研究结果表明微囊藻毒素LR可引起多种细胞发生凋亡[5],本实验室先前的研究也发现微囊藻毒素LR能激活在凋亡过程中起重要作用的酶Caspase-3[6],及引起P53、Bax、Bcl-2等凋亡相关蛋白…     

群体和单细胞微囊藻对短期高光胁迫的生理响应

为了探讨不同形态的微囊藻（Microcystis）对光的耐受能力及其应对机制,研究比较了短期高光强条件下群体微囊藻和单细胞微囊藻的生理响应,结果表明,在高光强胁迫下,群体和单细胞微囊藻的叶绿素含量、最大电子传递速率（ETR_max）均降低,但与单细胞微囊藻相比,群体微囊藻的下降幅度较小;在高光强胁迫下,群体微囊藻的过氧化氢酶（CAT）与超氧化物歧化酶（SOD）的活性均显著增加,而单细胞微囊藻只有CAT活性增加;在短期高光胁迫下,群体微囊藻的死亡率没有显著变化。这些结果表明群体微囊藻比单细胞微囊藻能耐受更高的光强,也暗示了群体微囊藻在野外高光强条件下更具竞争优势。     

群体和单细胞微囊藻对短期温度变化的生理响应

为了探索短期温度变化对群体微囊藻和单细胞微囊藻的影响, 在室内受控模拟条件下研究了在10℃、25℃和35℃三个温度梯度下, 群体和单细胞微囊藻对短期温度变化的生理响应。研究表明: 与对照组25℃相比, 在10℃培养下, 微囊藻叶绿素浓度显著降低, SOD活性和死亡率均显著增加。与群体微囊藻相比, 在10℃下单细胞微囊藻叶绿素浓度显著下降, F_v/F_m下降, SOD活性显著增加。在35℃培养下, 单细胞微囊藻叶绿素浓度上升, 死亡率和SOD活性增加, 而群体微囊藻则呈现出叶绿素浓度和死亡率降低, CAT活性增加。结果表明短期的温度变化影响了群体和单细胞微囊藻生理机制, 与单细胞微囊藻相比, 群体更能适应短期的温度胁迫, 导致其更具优势。     

Distribution and toxicity of a new colonial Microcystis aeruginosa bloom in the San Francisco Bay Estuary,California

The first distribution, biomass and toxicity study of a newly established bloom of the colonial cyanobacteria Microcystis aeruginosa was conducted on October 15, 2003 in the upper San Francisco Bay Estuary. Microcystis aeruginosa was widely distributed throughout 180 km of waterways in the upper San Francisco Bay Estuary from freshwater to brackish water environments and contained hepatotoxic microcystins at all stations. Other cyanobacteria toxins were absent or only present in trace amounts. The composition of the microcystins among stations was similar and dominated by demethyl microcystin-LR followed by microcystin-LR. In situ toxicity computed for the >75 m cell diameter size fraction was well below the 1 g l^–1 advisory level set by the World Health Organization for water quality, but the toxicity of the full population is unknown. The toxicity may have been greater earlier in the year when biomass was visibly higher. Toxicity was highest at low water temperature, water transparency and salinity. Microcystins from the bloom entered the food web and were present in both total zooplankton and clam tissue. Initial laboratory feeding tests suggested the cyanobacteria was not consumed by the adult copepod Eurytemora affinis, an important fishery food source in the estuary.     

短期淹水培养对水稻土中地杆菌和厌氧粘细菌丰度的影响

模拟水稻土淹水过程,采用Real-time PCR技术测度了不同水稻土中地杆菌(Geobacteraceae spp.)和厌氧粘细菌(Anaeromyxobacter spp.)在不同淹水时期16S rDNA拷贝数的变化,比较了地杆菌和厌氧粘细菌丰度与培养过程中微生物Fe (Ⅲ)还原的关系。结果表明,在4类稻作区采集的水稻土样品中,Fe (Ⅲ)还原潜势有明显的区别,表现出由北向南逐渐降低的趋势。从淹水12 h的地杆菌和厌氧粘细菌拷贝数变化看出,采自浙江和天津的水稻土样品对淹水过程具有高度敏感性,而采自吉林和广西的水稻土样品对淹水响应不敏感。在17 d的短期淹水培养中,地杆菌丰度明显高于厌氧粘细菌,表明地杆菌对水稻土中铁还原的贡献大于厌氧粘细菌。地杆菌和厌氧粘细菌拷贝数总体上表现出在11 d 时达到峰值,17 d时显著下降。吉林水稻土中地杆菌丰度在5 d时达到38.3%,与其最大铁还原速率到达时间(T_Vmax)为4.07 d相对应,表明地杆菌对其铁还原过程具有重要贡献。四川水稻土中地杆菌和厌氧粘细菌丰度均较低,暗示其他兼性铁还原菌对其铁还原的作用值得重视。     

The Tissue Distribution and Developmental Changes of ghrelin mRNA Expression in Sheep

Male Kazak sheep and Xinjiang fine wool sheep, six for each different age group (days 2, 30, 60, 90 and 120), were used in the present study to investigate the tissue distribution and developmental changes of ghrelin mRNA expression in abomasum; however, there was no 120-day-old Kazak sheep. After measurement of body weight, the tissues such as hypothalamus, pituitary, heart, liver, rumen, reticulum, omasum, abomasum, duodenum, and longissimus dorsi muscle were sampled. And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR and real-time PCR. The results showed that (1) for both breeds, body weight among different ages was significantly different (P<0.05). And from day 30 to 90, the body weight of Kazak was significantly higher than that of Xinjiang (P<0.01); (2) Ghrelin mRNA existed in all the above tissues and was significantly higher in the abomasum than in other tissues (P<0.05); (3) the temporal patterns of abomasum ghrelin mRNA expression in Kazak and Xinjiang were similar. From day 2 to 60 in Kazak and 2 to 90 in Xinjiang, there was a steady increase in the ghrelin mRNA level. By day 60 in Kazak and day 90 in Xinjiang, the level reached a plateau and remained steady. These results also demonstrated that from birth to day 90, ghrelin mRNA level was significantly higher in Kazak than in Xinjiang (P<0.01).     

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future.     

富营养化山仔水库沉积物微囊藻复苏的受控因子

山仔水库作为福建省福州市重要的饮用水水源地之一,从2000年起每年都周期性爆发蓝藻门微囊藻属(Microcystis)水华现象,特别是在温暖的季节。对于这个富营养化水库,是否在沉积物中存在蓝藻门微囊藻的\"种源\"？假设山仔水库底泥中存在蓝藻门微囊藻休眠体,一定的环境条件能够促进蓝藻门微囊藻的复苏。研究于2009年12月采集水库大坝断面5根柱状沉积物,采用正交试验的方法,模拟了温度、光照、pH值、营养盐、物理扰动和浮游动物(膨大肾形虫)等环境因子对山仔水库沉积物中蓝藻门微囊藻的复苏响应。结果表明,底泥中存在着一定数量的底栖动物和硅藻、蓝藻和绿藻等微藻,从实验结束后沉积物中微囊藻数量的减少和上覆水体中微囊藻数量的增加,可以判断在适宜的环境条件下,蓝藻门微囊藻能够复苏并上浮到上覆水体中。正交实验显著性分析表明,温度是沉积物蓝藻门微囊藻复苏的重要影响因子,光照次之,上覆水体的pH值、营养盐、物理扰动和浮游动物干扰对沉积物蓝藻门微囊藻的复苏影响作用不显著,升温有利于沉积物中微囊藻的复苏。     

Entomopathogenic nematodes, phoretic Paenibacillus spp., and the use of real time quantitative PCR to explore soil food webs in Florida citrus groves

Quantitative real-time PCR (qPCR) is a powerful tool to detect and quantify species of cryptic organisms such as bacteria, fungi and nematodes from soil samples. As such, qPCR offers new opportunities to study the ecology of soil habitats by providing a single method to characterize communities of diverse organisms from a sample of DNA. Here we describe molecular tools to detect and quantify two bacteria (Paenibacillus nematophilus and Paenibacillus sp.) phoretically associated with entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematodae. We also extend the repertoire of species specific primers and TaqMan® probes for EPNs to include Heterorhabditis bacteriophora, Steinernema carpocapsae, Steinernema feltiae and Steinernema scapterisci, all widely distributed species used commercially for biological control. Primers and probes were designed from the ITS rDNA region for the EPNs and the 16S rDNA region for the bacteria. Standard curves were established using DNA from pure cultures of EPNs and plasmid DNA from the bacteria. The use of TaqMan probes in qPCR resolved the non-specificity of EPN and some bacterial primer amplifications whereas those for Paenibacillus sp. also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two species that are not phoretically associated with nematodes. The primer-probe sets for EPNs were able to accurately detect three infective juvenile EPNs added to nematodes recovered from soil samples. The molecular set for Paenibacillus sp. detected the bacterium attached to Steinernema diaprepesi suspended in water or added to nematodes recovered from soil samples but its detection decreased markedly in the soil samples, even when a nested PCR protocol was employed. Using qPCR we detected S. scapterisci at low levels in a citrus grove, which suggested natural long-distance spread of this exotic species, which is applied to pastures and golf courses to manage mole crickets (Scapteriscus spp.). Paenibacillus sp. (but not P. nematophilus) was detected in low quantities in the same survey but was unrelated to the spatial pattern of S. diaprepesi. The results of this research validate several new tools for studying the ecology of EPNs and their phoretic bacteria.